The purpose of this study was to evaluate the role of nitric oxide (NO) in the maintenance of basal endometrial blood flow of ovariectomized rats and in the increase of endometrial blood flow after administration of estradiol 17 (E₂). Endometrial blood flow was repeatedly measured with the H₂ gas clearance technique in ovariectomized rats. N-nitro-L-arginine methyl ester (L-NAME) dose dependently reduced basal endometrial blood flow and increased mean arterial blood pressure and endometrial vascular resistance. E₂ (1 µg/kg iv) increased endometrial blood flow and reduced endometrial vascular resistance, which peaked by 2 h after the injection. The vasoconstrictive activity of L-NAME (an inhibitor for NO synthesis) was compared with that of phenylephrine (PE, an -receptor agonist acting through an NO-independent mechanism). Doses of L-NAME (1 and 3 mg/kg iv) were matched with those of PE (3.2 and 6.4 mg · kg¹ · h¹ iv), as they induced an approximately equivalent percent increase in basal endometrial vascular resistance. The percent increases of endometrial vascular resistance in E₂-treated animals by the two agents in matched doses were also of a similar magnitude. When animals were first treated with L-NAME or PE, E₂ lost the ability to reduce endometrial vascular resistance. Enzyme activity and gene expression of NO synthase in the rat uterine tissue were also examined after E₂ treatment, and no significant changes were observed. These data raise doubts about the role of NO in the regulation of endometrial blood flow after acute administration of E₂ and suggest that other mechanisms may be involved.